This invention relates to a biological activity probe for the detection on-line of the presence of sulphide. It is thus applicable for the detection of a level of microbiological activity involving sulphide which is likely to influence corrosion, for example of a buried metal pipeline, or to result in the formation of biofilm surface coatings, for example inside either metallic or non-metallic pipes. The probe is thus of use in monitoring biological activity both inside and outside a number of structures, such as buried pipelines, and heat exchanger structures.
Since the discovery of sulphate reducing bacteria by Bijerninck in 1905, microbiologically influenced corrosion has been the subject of considerable interest. Microbiologically influenced corrosion has been documented for metals, particularly ferrous metals, exposed to seawater, fresh water, water associated with crude oil and with natural gas, demineralised water, chemical process flows, foods, aircraft fuels, human plasma, and sewage. Despite the evidence that the presence of a number of different groups of microorganisms can influence and accelerate rates of corrosion, sulphide producing microorganisms, including sulphate reducing bacteria (SRB), have received the most attention as the causative, agents of microbiologically influenced corrosion for a number of reasons. In addition to being the most common causative group of microorganisms involved in microbiologically influenced corrosion, they were identified as being involved in the first corrosion mechanism that accounted for anaerobic corrosion of ferrous metals. In a suitable environment, sulphate reducing bacteria will produce sulphide ions, typically as hydrogen sulphide, which will corrode metals, including ferrous metals, copper, and nickel alloys.
A parallel problem resulting from the presence of active bacteria is the formation of adherent coatings of biofilm on a wide variety of surfaces, which includes the surfaces of both metallic and non-metallic materials, such as the inside of metal and plastic pipes. The formation of an internal biofilm obstructs the pipe bore and thus impairs its flow capacity, and in applications such as heat exchangers can significantly degrade pipe heat exchange properties.
Although the involvement of sulphide producing microorganisms (e.g. SRB) in microbiologically influenced corrosion is well known, and conditions have been identified that are suitable for the growth of sulphide producing bacteria, analytical methods are still lacking which will provide definitive indicators that microbiologically influenced corrosion in fact will occur. For example, there does not appear to be any consistent correlation between the size of a population of sulphate reducing bacteria present in a given environment, and the microbiologically influenced corrosion in that environment. Further, at present there are no definitive tests available that can be used on line for a structure in the field to detect microbiologically influenced corrosion, although there are some features of the results of microbiologically influenced corrosion which have been identified, such as non-uniform pitting, pits filled with black corrosion products, round pits under tubercles in carbon steel, and pinholes leading to large subsurface cavities in stainless steel. Consequently, in the absence of definitive and reliable indicators, identification of the presence of conditions which will foster microbiologically influenced corrosion is difficult.
In the past, three approaches have been proposed as a means to monitor conditions which can be taken to indicate that microbiologically influenced corrosion might be occurring. These are to monitor the relative quantity of microorganisms that are present, to monitor microbiological influences, and to monitor on-going corrosion.
In order to monitor the number of organisms present, techniques utilising more or less conventional culturing procedures have been used, with both an iron source and sulphate ions being included in the culture medium. If sulphate reducing bacteria are present, black ferrous sulphide is formed. The number of positive samples in a most probable number method can be used to assess the quantity of sulphate reducing bacteria that are present. Alternatively, several direct methods for detecting the presence of sulphide producing microorganisms have been proposed. In these methods, the method does not require the growth of microorganisms during the test. Instead, after removal of dissolved solids that might interfere with the test, a photochemical procedure, usually involving enzymatic reduction, is used.
In order to monitor microbiological influences, several indirect methods have been proposed. The presence of an adhering biofilm on a metallic surface alters the properties of that surface, and can also modify the local anodic and cathodic processes. For example, the simple presence of a biofilm will alter the pressure drop through an orifice, and will alter the heat transfer properties of a tube surface, provided that there is enough biofilm formation to provide meaningful results.
In order to monitor on-going corrosion, several procedures have been proposed, which seek to provide an alert that the metallic system is corroding, and at what rate. Commonly used methods include weight loss coupons, galvanic probes, electrochemical probes and system simulations.
These proposed systems all however suffer from several significant disadvantages.
Although the biological procedures are capable of indicating that sulphide producing microorganisms are present, and often an indication of the relative size of the sampled population, these methods give no information at all as to whether microbiologically influenced corrosion might be actually happening. Furthermore, the biological procedures are only useable on a relatively small scale and in a laboratory environment; they are not capable of being used on line in the field to monitor any activity in the environment of a structure.
Although the measurements of microbiological presence are sometimes capable of detecting physical changes, such as the consequences of biofilm formation, these methods too do not indicate whether microbiologically influenced corrosion is either likely to happen or even happening. For example, a reduction in the heat transfer properties of a tube only indicates that a biofilm is likely to be present, and cannot give any useful information about the bacteria in that biofilm.
Although corrosion monitoring methods provide information on total corrosion rates, these methods do not provide any information on either whether the corrosion includes any microbiologically influenced corrosion, or the relative rate of any microbiologically influenced corrosion if it is occurring.
Thus although microbiologically influenced corrosion has been invoked as the cause of many unexpected corrosion failures, typically of buried steel pipes, evidence identifying microbiological activity as the primary cause of the failure continues to be elusive. There is no currently available technique that can be used to characterise a microbial population in the field, and that can provide useful information about the potential microbiologically influenced corrosion risks to which a given structure is exposed.
This invention seeks to provide a biological activity probe for monitoring microbiological activity in the environment of a structure, to detect the presence of microorganisms which to produce substances such as sulphides which are known to have a direct influence on corrosion. The probe of this invention can be used to detect the activity of sulphide producing microorganisms (e.g. SRB""s ) both in situ and on line on a real time basis. In a preferred embodiment, the probe can provide information on corrosion conditions. The probe can be used to detect the activity of microorganisms such as anaerobic sulphide producing microorganisms e.g. SRB), and is believed to function also under aerobic conditions.
Thus in a first embodiment this invention seeks to provide a probe for monitoring the activity of sulphide producing microorganisms including:
(1) an enzyme electrode;
(2) a counter electrode; and
(3) a reference electrode;
which are all connectable to a suitable electrical system for data acquisition, wherein:
the enzyme electrode comprises an immobilised source of sulfide oxidase enzyme, together with a cofactor, immobilised in a water permeable non-conducting binder on a biologically inert electrically conducting substrate;
the cofactor comprises an artificial mediator that replaces oxygen under anaerobic conditions;
the counter electrode comprises a biologically inert electrically conducting electrode; and
the reference electrode comprises a biologically inert electrically conducting electrode.
In a second embodiment this invention seeks to provide a probe for monitoring the activity of sulphide producing microorganisms in the environment of a structure incorporating a metal including:
(1) an enzyme electrode;
(2) a counter electrode;
(3) a reference electrode; and
(4) a corrosion electrode;
which are all connectable to a suitable electrical system for data acquisition, wherein:
the enzyme electrode comprises an immobilised source of sulfide oxidase enzyme, together with a cofactor, immobilised in a water permeable non-conducting binder on a biologically inert electrically conducting substrate;
the cofactor comprises an artificial mediator that replaces oxygen under anaerobic conditions;
the counter electrode comprises a biologically inert electrically conducting electrode;
the reference electrode comprises a biologically inert electrically conducting electrode; and
the corrosion electrode comprises a metal electrode.
Preferably, the immobilised source of sulphide oxidase enzyme comprises a chemoautotropic microorganism. Preferably, the chemoautotropic microorganism is a Thiobacillus thioparus species. Alternatively, the immobilised source of sulphide oxidase enzyme comprises enzymatic material recovered from a culture of a Thiobacillus thioparus species.
Preferably, the cofactor is chosen from the group consisting of pyocyanine, phenazine metasulphate, 1,2-naphthoquinone, 2,6-dichlorophenolindophenol, tetracyanoquinodimethane, 1,4-benzoquinone, the compound Ru(NH3)5pyridinium(PF6), chlornil, potassium ferricyanide, N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-p-phenylenediamine, ferrocene monocarboxylic acid, tetrathiafulvene, ferrocene and 1,1xe2x80x2-dimethyl ferrocene. More preferably, the cofactor is chosen from 1,1xe2x80x2-dimethylferrocene and ferrocene. Most preferably, the cofactor is 1,1xe2x80x2-dimethylferrocene.
Conveniently, the counter electrode, the reference electrode and the corrosion electrode if present are the same.
Preferably, the counter electrode is chosen from the group consisting of a graphite electrode, a stainless steel electrode, and a steel electrode.
Preferably, the reference electrode is chosen from the group consisting of a graphite electrode, a stainless steel electrode, a carbon steel electrode; a standard calomel electrode (SCE), a copper/copper sulphate electrode, and a silver/silver chloride electrode.
Preferably, the electrodes are mounted within a protective outer shell having an opening at its first end for the electrode active surfaces, and having provision for electrical connections to the electrodes at its second end, and the electrodes are separated from each other within the shell by a biologically inert insulating material.
The probe of this invention thus relies on the enzymatic oxidation of sulphide, which requires the presence of a sulphur oxidase enzyme. In the context of a probe, this can be achieved in two ways: either the probe includes a suitable amount of an immobilised chemoautotropic microorganism, such as a Thiobacillus thioparus, or the probe includes a suitable amount of a sulphur oxidase enzyme, such as an active enzyme concentrate derived from a Thiobacillus thioparus. For each of these options the construction of the probe for either laboratory or for field use is more or less the same, the chief differences being the active material which is immobilised on the enzyme electrode, and the technique used to prepare the active material. For either option, a suitable microorganism source material is required.